High-throughput Neutralization Research Articles

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains.

We describe a new approach that can rapidly measure how the antibodies in human serum inhibit infection by many different influenza strains. This new approach is useful for understanding how viral evolution affects antibody immunity. We apply the approach to study the effect of repeated influenza vaccination.

Optimization of Cellular Transduction by the HIV-Based Pseudovirus Platform with Pan-Coronavirus Spike Proteins.

Over the past three years, new SARS-CoV-2 variants have continuously emerged, evolving to a point where an immune response against the original vaccine no longer provided optimal protection against these new strains. During this time, high-throughput neutralization assays based on pseudoviruses have become a valuable tool for assessing the efficacy of new vaccines, screening updated vaccine candidates against emerging variants, and testing the efficacy of new therapeutics such as monoclonal antibodies. Lentiviral vectors derived from HIV-1 are popular for developing pseudo and chimeric viruses due to their ease of use, stability, and long-term transgene expression. However, the HIV-based platform has lower transduction rates for pseudotyping coronavirus spike proteins than other pseudovirus platforms, necessitating more optimized methods. As the SARS-CoV-2 virus evolved, we produced over 18 variants of the spike protein for pseudotyping with an HIV-based vector, optimizing experimental parameters for their production and transduction. In this article, we present key parameters that were assessed to improve such technology, including (a) the timing and method of collection of pseudovirus supernatant; (b) the timing of host cell transduction; (c) cell culture media replenishment after pseudovirus adsorption; and (d) the centrifugation (spinoculation) parameters of the host cell+ pseudovirus mix, towards improved transduction. Additionally, we found that, for some pseudoviruses, the addition of a cationic polymer (polybrene) to the culture medium improved the transduction process. These findings were applicable across variant spike pseudoviruses that include not only SARS-CoV-2 variants, but also SARS, MERS, Alpha Coronavirus (NL-63), and bat-like coronaviruses. In summary, we present improvements in transduction efficiency, which can broaden the dynamic range of the pseudovirus titration and neutralization assays.

Virus neutralization assays for human respiratory syncytial virus using airway organoids

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.

Longitudinal and Cross-Sectional Evaluation of Two Commercial Swine Breeding Herds to Characterize Neutralizing Antibody Levels following Porcine Epidemic Diarrhea Virus Outbreaks.

Neutralizing antibodies to Porcine Epidemic Diarrhea Virus (PEDV) can be detected by 3 weeks post-infection and remain detectable through at least 24 weeks post-infection. The objective of this study was to evaluate the levels of neutralizing antibodies in sow and piglet serum and sow milk to determine the duration of neutralizing antibodies following PEDV outbreaks. Two farms were selected for the study following outbreaks of PEDV. Monthly, cohorts of sows were sampled and followed through two farrowings. Following each farrowing, samples from piglets and milk were collected. Samples were evaluated for PEDV-neutralizing antibodies by a high-throughput fluorescent neutralization assay. Although neutralizing antibodies to PEDV can be detected throughout 15 months post-outbreak, a decrease in circulating neutralizing antibody levels is noted in farms beginning at six months post-outbreak. With decreasing levels, farms may become more vulnerable to PEDV outbreaks, and practitioners can focus on this time window to implement intervention strategies.

The development of a rapid, high-throughput neutralization assay using a SARS-CoV-2 reporter

Many methods have been developed to measure the neutralizing capacity of antibodies to SARS-CoV-2. However, these methods are low throughput and can be difficult to quickly modify in response to emerging variants. Therefore, an experimental system for rapid and easy measurement of the neutralizing capacity of antibodies against various variants is needed. In this study, we developed an experimental system that can efficiently measure the neutralizing capacity of sera by using a GFP-carrying recombinant SARS-CoV-2 with spike proteins of multiple variants (B.1.1, BA.5, or XBB.1.5). For all 3 recombinant chimeric genomes generated, neutralizing antibody titers determined by measuring GFP fluorescence intensity correlated significantly with those calculated from viral RNA levels measured by RT-qPCR in the supernatant of infected cells. Furthermore, neutralizing antibody titers determined by visually assessing GFP fluorescence using microscopy were also significantly correlated with those determined by RT-qPCR. By using this high-throughput method, it is now possible to quickly and easily determine the neutralizing capacity of antibodies against SARS-CoV-2 variants.

Development and characterization of a recombinant Senecavirus A expressing enhanced green fluorescent protein.

Senecavirus A (SVA), belonging to the genus Senecavirus in the family Picornaviridae, is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development. In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes. We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. In mice, rSVA-eGFP also showed similar replication kinetics and pathogenicity to the parental virus, both causing mild lung lesions. In addition, a high-throughput viral neutralization assay was developed using eGFP as a surrogate readout in a fluorescence-based direct titration (FBT) assay based on rSVA-eGFP, facilitating rapid and accurate determination of the neutralizing antibody (nAb) titers. The successful establishment of an SVA reverse genetics system and the rescue of rSVA-eGFP would create a powerful tool for future studies of SVA replication mechanisms and pathogenicity as well as for antiviral development.

Advances on two serological assays for human papillomavirus provide insights on the reactivity of antibodies against a cross-neutralization epitope of the minor capsid protein L2.

A second generation of prophylactic human papillomavirus (HPV) vaccines based on the minor capsid protein L2 has entered clinical trials as promising alternative to meet the gaps left out by the current vaccines concerning type-restricted protection, high costs and low penetrance in immunization programs of lowand middle-income countries. Most of the serological assays available to assess anti-HPV humoral responses are, however, not well suited for measuring vaccine-induced anti-L2 antibody responses. In this work, we have advanced our automated, purely add-on High-Throughput Pseudovirion-Based Neutralization Assay (HT-PBNA) in an L2-oriented approach for measuring antibody-mediated neutralization of HPV types 6/16/18/31/33/52/58. With the optimized settings, we observed 24- to 120-fold higher sensitivity for detection of neutralizing Ab to the L2 protein of HPV6, HPV16, HPV18, and HPV31, compared to the standard HT-PBNA. Alternatively, we have also developed a highly sensitive, cell-free, colorimetric L2-peptide capture ELISA for which the results were strongly concordant with those of the advanced neutralization assay, named HT-fc-PBNA. These two high-throughput scalable assays represent attractive approaches to determine antibody-based correlates of protection for the HPV L2 vaccines that are to come.

Sars-Cov-2 Antibody and T-Cell Responses in Patients with Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia after Four COVID-19 Vaccinations

Introduction: There is limited data on SARS-CoV-2 vaccination responses in the rare hematological disorders paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA). These patients were expected to have more severe COVID-19 infection and reduced vaccination responses due to their underlying disease and immunosuppressive treatment. We previously reported limited anti-spike IgA/G/M responses after first COVID-19 vaccination which improved following second vaccination (Pike et al, Lancet Haematology 2022). Here we report spike-specific IgG, in vitro viral neutralization and T-cell responses in 244 patients with PNH and/or AA and in 49 healthy volunteers, following the first four vaccinations. Methods: In 2021, the United Kingdom National PNH service centre based in Leeds initiated a prospective observational non-interventional study evaluating immune responses to COVID-19 vaccinations in patients with PNH and/or AA. All patients were consented to the Leeds PNH Research Tissue Bank. At baseline, the patient cohort comprised 94 patients with classic PNH, 75 with AA-PNH overlap and 75 with AA with asymptomatic PNH clones <50%. Serological humoral responses were evaluated in blood samples using a quantitative spike-specific IgG SARS-CoV-2 ELISA (EuroImmun). Samples were further analyzed using a high-throughput live-virus neutralization assay performed at the Francis Crick Institute, London, against wild-type SARS-CoV-2 and against omicron B.1.1.529 and delta B.1.617.2 variants. Adaptive T-cell immune responses were assessed by incubating cryopreserved peripheral blood mononuclear cells with SARS-CoV-2 spike and nucleocapsid peptides in an ELISpot interferon-gamma release assay (Oxford Immunotec). Results: Prior to vaccination (baseline) in-vitro anti-spike IgG response was not significantly different between patients and healthy volunteers (p>0.99). After first vaccination, 2/40 (5%) healthy volunteers and 66/155 (42.6%) PNH and AA patients failed to mount a detectible spike-specific IgG. The antibody titre was also significantly reduced (median spike-specific IgG titre in patients 37.9 BAU/ml [IQR 11.56-120.7] versus 289.4 BAU/ml in healthy volunteers [IQR 177.7-1326], p<0.0001). The magnitude of IgG response improved after second vaccination but remained lower than healthy volunteers (median IgG titre in patients 408.6 BAU/ml [IQR 184.1-942.6] versus healthy volunteers 2639 BAU/ml [IQR 894.0-3706], p<0.001). However, responses improved sequentially following repeated vaccinations and after the third dose were equivalent to healthy volunteers (p>0.99). Viral neutralizing antibody activity elicited by vaccination showed similar results, with significantly reduced titres post first vaccination against wild-type SARS-CoV-2 in patients compared with healthy volunteers (p=0.0001). After second vaccination, neutralizing antibody titres in patients versus healthy volunteers remained significantly reduced against wild-type (p=0.0088) and the delta variant (p=0.0001) but not the omicron variant (p=0.078). Reassuringly, responses improved after 3 rd dose of vaccine following which there was no significant difference detected for all three strains (p>0.99). Of the evaluable samples, anti-spike T-cell responses were reduced at all timepoints in patients compared with healthy volunteers but improved with each vaccination. After first vaccination, a reactive result was seen in 32.1% (n=27/84) versus 40% in healthy volunteers (n=16/40) and remained low after second vaccination (35.0% (n=43/123) in patients versus 64.1% (n=24/39) in healthy volunteers). However, after third vaccination, responses had improved to 58.7% (n=44/75) in patients versus 75% (n=24/32) in healthy volunteers. After 4 th vaccination, spike-specific responses were detectable in 67.6% (n=25/37) of patients. There was limited reactivity to nucleocapsid antigens in both groups at all timepoints highlighting that the observed responses were due to vaccination rather than COVID-19 infection. To date there have been no deaths due to COVID-19 infection in patients in this study or in our wider service population since the introduction of the vaccination programme. Conclusion: This study demonstrates the importance of repeated SARS-Cov-2 vaccinations in patients with PNH and/or AA in order to improve humoral and cellular immune responses.

A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay.

Despite the success of rotavirus vaccines, rotaviruses remain one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality, especially in low- and middle-income countries. The reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins, such as vaccine antigens and therapeutic payloads. Here, we demonstrate that three positions in rotavirus genome-the C terminus of NSP1, NSP3 and NSP5-can tolerate the insertion of reporter genes. By using rotavirus expressing GFP, we develop a high-throughput neutralization assay and reveal the pre-existing immunity against rotavirus in humans and other animal species. Our work shows the plasticity of the rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.

Development of an automated, high-throughput SARS-CoV-2 neutralization assay based on a pseudotyped virus using a vesicular stomatitis virus (VSV) vector

ABSTRACT The global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293 T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z’ factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.

Cross-sectional Characterization of SARS-CoV-2 Antibody Levels and Decay Rates Following Infection of Unvaccinated Elderly Individuals.

SARS-CoV-2 infections have disproportionally burdened elderly populations with excessive mortality. While several contributing factors exists, questions remain about the quality and duration of humoral antibody-mediated responses resulting from infections in unvaccinated elderly individuals. Residual serum/plasma samples were collected from individuals undergoing routine SARS-CoV-2 polymerase chain reaction testing in a community laboratory in Canada. The samples were collected in 2020, before vaccines became available. IgG, IgA, and IgM antibodies against SARS-CoV-2 nucleocapsid, trimeric spike, and its receptor-binding domain were quantified via a high-throughput chemiluminescent enzyme-linked immunosorbent assay. Neutralization efficiency was also quantified through a surrogate high-throughput protein-based neutralization assay. This study analyzed SARS-CoV-2 antibody levels in a large cross-sectional cohort (N = 739), enriched for elderly individuals (median age, 82 years; 75% >65 years old), where 72% of samples tested positive for SARS-CoV-2 by polymerase chain reaction. The age group ≥90 years had higher levels of antibodies than that <65 years. Neutralization efficiency showed an age-dependent trend, where older persons had higher levels of neutralizing antibodies. Antibodies targeting the nucleocapsid had the fastest decline. IgG antibodies targeting the receptor-binding domain remained stable over time, potentially explaining the lack of neutralization decay observed in this cohort. Despite older individuals having the highest levels of antibodies postinfection, they are the cohort in which antibody decay was the fastest. Until a better understanding of correlates of protection is acquired, along with the protective role of nonneutralizing antibodies, booster vaccinations remain important in this demographic.

Comparison of Two Diagnostic Assays for the Detection of Serum Neutralizing Antibody to Porcine Epidemic Diarrhea Virus

Lactogenic immunity is important for the protection of piglets against many pathogens including porcine epidemic diarrhea virus. Circulating neutralizing antibodies levels in sow sera may help determine if a detectable immune response could confer protection to piglets. Neutralizing antibodies can be detected through various diagnostic assays. This study evaluated the diagnostic characteristics of two neutralizing antibody assays for porcine epidemic diarrhea virus neutralizing antibodies in serum of challenged gilts. Four treatment groups, control, non-vaccinated, vaccinated prior to challenge, and vaccinated following challenge, were comprised of 20 gilts. Serum sample were collected from each gilt prior to and following challenge with porcine epidemic diarrhea virus. Samples were evaluated for the presence of neutralizing antibodies via a fluorescent focus neutralization assay and a high-throughput neutralization assay. Diagnostic sensitivity and specificity for the fluorescent focus neutralization and high-throughput neutralization assays for this study were optimized at a cutoff of a dilution of 80 and 80% fluorescent reduction respectively and demonstrated moderate agreement based off the kappa statistic. The focus fluorescent neutralization and high-throughput neutralization assays can be used to monitor the status of neutralizing antibodies within animals or a population of animals. The high-throughput assay has advantages over the focus fluorescent assay in that it has a higher specificity at the indicated cut-off and the nature of the results allows for more discrimination between individual results.

Trajectories of Seroprevalence and Neutralizing Activity of Antibodies against SARS-CoV-2 in Southern Switzerland between July 2020 and July 2021: An Ongoing, Prospective Population-Based Cohort Study.

The COVID-19 pandemic continues, and evidence on infection- and vaccine-induced immunity is key. We assessed COVID-19 immunity and the neutralizing antibody response to virus variants across age groups in the Swiss population. We conducted a cohort study in representative community-dwelling residents aged five years or older in southern Switzerland (total population 353,343), and we collected blood samples in July 2020 (in adults only, N = 646), November-December 2020 (N = 1457), and June-July 2021 (N = 885). We used a previously validated Luminex assay to measure antibodies targeting the spike (S) and the nucleocapsid (N) proteins of the virus and a high-throughput cell-free neutralization assay optimized for multiple spike protein variants. We calculated seroprevalence with a Bayesian logistic regression model accounting for the population's sociodemographic structure and the test performance, and we compared the neutralizing activity between vaccinated and convalescent participants across virus variants. The overall seroprevalence was 7.8% (95% CI: 5.4-10.4) by July 2020 and 20.2% (16.4-24.4) by December 2020. By July 2021, the overall seroprevalence increased substantially to 72.5% (69.1-76.4), with the highest estimates of 95.6% (92.8-97.8) among older adults, who developed up to 10.3 more antibodies via vaccination than after infection compared to 3.7 times more in adults. The neutralizing activity was significantly higher for vaccine-induced than infection-induced antibodies for all virus variants (all p values < 0.037). Vaccination chiefly contributed to the reduction in immunonaive individuals, particularly those in older age groups. Our findings on the greater neutralizing activity of vaccine-induced antibodies than infection-induced antibodies are greatly informative for future vaccination campaigns.

High-Throughput Neutralization and Serology Assays Reveal Correlated but Highly Variable Humoral Immune Responses in a Large Population of Individuals Infected with SARS-CoV-2 in the US between March and August 2020.

The ability to measure neutralizing antibodies on large scale can be important for understanding features of the natural history and epidemiology of infection, as well as an aid in determining the efficacy of interventions, particularly in outbreaks such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Because of the assay's rapid scalability and high efficiency, serology measurements that quantify the presence rather than function of serum antibodies often serve as proxies of immune protection. Here, we report the development of a high-throughput, automated fluorescence-based neutralization assay using SARS-CoV-2 virus to quantify neutralizing antibody activity in patient specimens. We performed large-scale testing of over 19,000 COVID-19 convalescent plasma (CCP) samples from patients who had been infected with SARS-CoV-2 between March and August 2020 across the United States. The neutralization capacity of the samples was moderately correlated with serological measurements of anti-receptor-binding domain (RBD) IgG levels. The neutralizing antibody levels within these convalescent-phase serum samples were highly variable against the original USA-WA1/2020 strain with almost 10% of individuals who had had PCR-confirmed SARS-CoV-2 infection having no detectable antibodies either by serology or neutralization, and ~1/3 having no or low neutralizing activity. Discordance between neutralization and serology measurements was mainly due to the presence of non-IgG RBD isotypes. Meanwhile, natural infection with the earliest SARS-CoV-2 strain USA-WA1/2020 resulted in weaker neutralization of subsequent B.1.1.7 (alpha) and the B.1.351 (beta) variants, with 88% of samples having no activity against the BA.1 (omicron) variant. IMPORTANCE The ability to directly measure neutralizing antibodies on live SARS-CoV-2 virus in individuals can play an important role in understanding the efficacy of therapeutic interventions or vaccines. In contrast to functional neutralization assays, serological assays only quantify the presence of antibodies as a proxy of immune protection. Here, we have developed a high-throughput, automated neutralization assay for SARS-CoV-2 and measured the neutralizing activity of ~19,000 COVID-19 convalescent plasma (CCP) samples collected across the United States between March and August of 2020. These data were used to support the FDA's interpretation of CCP efficacy in patients with SARS-CoV-2 infection and their issuance of emergency use authorization of CCP in 2020.

Development of rapid neutralization assay of viral hemorrhagic septicemia virus (VHSV) based on chimeric rhabdovirus expressing heterologous glycoprotein

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV.

Development of a rapid neutralization testing system for Rhinovirus C15 based on the enzyme-linked immunospot assay.

Human Rhinoviruses (RVs) are dominant pathogens causing a wide range of respiratory tract diseases, posing a huge threat to public health worldwide. Viruses belonging to the RV-C species are more likely to cause severe illnesses and are strongly associated with asthma onset or exacerbations than RV-A or RV-B. Rapid and sensitive detection of neutralizing antibodies (NAbs) against RV-C can promote the development of vaccines and antiviral drugs and help in the diagnosis of viral infection. In this study, a rapid neutralization testing system for RV-C15, based on an enzyme-linked immunospot assay (Nt-ELISPOT) was developed. A monoclonal antibody (MAb), named 9F9, with high binding efficacy for RV-C15 conjugated to horseradish peroxidase (HRP), was used to detect RV-C15-infected cells at a concentration of 2 μg/ml. The optimal infectious dose of RV-C15 was set at 1 × 104 TCID50/well and the cells were fixed with 0.5% formaldehyde diluted in PBS after incubation for 20 h. Compared with the traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE), Nt-ELISPOT significantly shortened the detection period and showed good consistency with the detection of neutralizing titers of both sera and NAbs. Using Nt-ELISPOT, three anti-RV-C15 NAbs were obtained with IC50 values of 0.16, 0.27, and 11.8 μg/ml, respectively. Moreover, 64 human serum samples collected from a wide range of age groups were tested for NAb against RV-C15 by Nt-ELISPOT. The total seroprevalence was 48.4% (31/64) and the positive rate was lowest in the group under 6 years old. Thus, the Nt-ELISPOT established in this study can be used as a high-throughput and rapid neutralization assay for the screening of NAbs and for seroepidemiological investigation against RV-C15.

A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions

We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcription regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (∆3678). The ∆3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The ∆3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the ∆3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the ∆3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter ∆3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that ∆3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.

A Single-Round Infection Fluorescent SARS-CoV-2 Neutralization Test for COVID-19 Serological Testing at a Biosafety Level-2 Laboratory.

A robust serological test to measure neutralizing antibodies against SARS-CoV-2 in biosafety level-2 (BSL-2) laboratories is useful for monitoring antibody response after vaccination or natural infection. The gold standard assay is the conventional plaque reduction neutralization test (PRNT) which requires extensive labor, live viruses, and BSL-3 facilities. Recently, we developed a novel single-round infection fluorescent SARS-CoV-2 virus (SFV) that can be safely used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. In this study, we evaluated the performance of the neutralization test using this SFV with 80 PRNT-positive and 92 PRNT-negative clinical serum or plasma specimens. The SFV neutralization test (SFVNT) has 100% sensitivity and specificity compared to the PRNT. Furthermore, the neutralizing titers generated by the SFVNT and PRNT are highly correlated, with R2 = 0.903 (p < 0.0001). Due to high sensitivity, specificity, accuracy, and reproducibility, the SFVNT can be deployed for the large-scale testing of COVID-19 patients or vaccinated people in general lab settings.

Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH

Human cytomegalovirus (HCMV) is a β-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

Neutralization against Omicron SARS-CoV-2 from previous non-Omicron infection

The spread of the Omicron SARS-CoV-2 variant underscores the importance of analyzing the cross-protection from previous non-Omicron infection. We have developed a high-throughput neutralization assay for Omicron SARS-CoV-2 by engineering the Omicron spike gene into an mNeonGreen USA-WA1/2020 SARS-CoV-2 (isolated in January 2020). Using this assay, we determine the neutralization titers (defined as the maximal serum dilution that inhibited 50% of infectious virus) of patient sera collected at 1- or 6-months after infection with non-Omicron SARS-CoV-2. From 1- to 6-month post-infection, the neutralization titers against USA-WA1/2020 decrease from 601 to 142 (a 4.2-fold reduction), while the neutralization titers against Omicron-spike SARS-CoV-2 remain low at 38 and 32, respectively. Thus, at 1- and 6-months after non-Omicron SARS-CoV-2 infection, the neutralization titers against Omicron are 15.8- and 4.4-fold lower than those against USA-WA1/2020, respectively. The low cross-neutralization against Omicron from previous non-Omicron infection supports vaccination of formerly infected individuals to mitigate the health impact of the ongoing Omicron surge.